PROJECT SUMMARY: More than 35.5 million people are living with AIDS as reported by World Health Organization (WHO) recently. HIV caused over 25 million deaths so far. Currently, only 34% of HIV infected people are getting antiretroviral therapy (ART) treatment in developing world despite ART being affordable or even freely available. The fundamental challenges to reduce HIV burden and its prevalence include the lack of point-of-care assays for viral load and CD4+ T cell count in rural areas lacking reliable electricity and refrigeration, and having limited access to basic laboratory infrastructure and trained technicians. The CD4+ T cell blood count has enormous prognostic and therapeutic implications, and forms the basis of HIV treatment decisions. According to current guidelines by WHO, HIV infected people with < 500 CD4+ T cells/l of whole blood should be treated with ART. In the developed world, CD4+ T cell counts rely on flowcytometer which is an expensive equipment. Further, a single CD4+ T cell count can cost upto $50 per test and requires technicians to perform test. Reagents used during flowcytometry need to be stored at low temperatures (4-8oC) to prevent the denaturation of antibodies and loss of their function. In addition to the issues related to cost around cold storage, whereas cold storage may not even be available in resource-limited settings. Due to these challenges, currently 1 in 4 people has CD4+ T cell count <100 cells/l at the start of ART treatment which reflects late diagnosis, and, hence, higher risk of illness and death. To address these challenges, there is an unmet need to develop a low- cost, reliable, label-free, and rapid CD4+ T cell counting technology that would not require trained technicians and the devices should be stable at refrigeration-free conditions. To meet these goals, we propose to develop a low-cost (<$1) transparency paper-based flexible microfluidic device that can efficiently isolates CD4+ T cells from a fingerprick of whole blood (<10l). The paper devices will be integrated with lensless imaging platform that provides ultra-wide field of view for rapid counting of all the captured cells in few seconds (~3 secs). We will also develop a novel protocol to stabilize the immune-functionalized microfluidic channels for storage at refrigeration-free conditions upto 12 months. These ready-to-use, disposable, and stabilized paper-based devices can be used at the point-of-need without any delay. We envision that the proposed microfluidic device will aid in rapid CD4+ T cell counting benefiting millions of patients around the world.